The parent proposal sponsors the study of the structure and mechanism of the oxygen-bridged diiron cluster-containing enzyme methane monooxygenase (MMO). Herewe propose a new aim in which the chemistry and regulation of MMO is compared with those of a previously unrecognized diiron monooxygenase family. We have purified the first member of this family (CmlA) from the biosynthetic pathway for chloramphenicol in Streptomyces, where it catalyzes ?-hydroxylation of L-p aminophenylalanine (PAPA). We have shown that both the steady state hydroxylation reaction and the single turnover reaction of the reduced diiron cluster of CmlA with O2 require interaction of CmlA with PAPA covalently loaded on the thiolation domain of a nonribosomal peptide synthetase (NRPS), CmlP. This differs from the O2 activation reaction of the hydroxylase component of MMO (MMOH) that occurs without substrate bound. The amino acid sequence of CmlA shows that it's C-terminal half aligns with the large family of metallo-?-lactamases that usually bind a di-zinc cluster. Nevertheless, metal analysis and EPR and M"ssbauer spectroscopic studies show unequivocally that CmlA binds a diiron cluster. This is the first example of an oxygen activating enzyme using this protein fold. The overall sequence of CmlA aligns with at least 50 uncharacterized enzymes that are part of the biosynthetic pathways for antibiotics and biostatics. We propose to: (i) use truncated CmlA and CmlP constructs to determine the minimal size proteins that can carry out O2 activation and hydroxylation, (ii) Use optical, EPR, and M"ssbauer spectroscopies to characterize the metal center of CmlA and structural perturbations that occur when it binds CmlP analogs, (iii) search for reaction cycle intermediates of CmlA using the single turnover system, and (iv) use diffracting single crystals to determine the X-ray crystal structure of CmlA. The markedly different protein environment and regulatory mechanism for the control of oxygen activation by CmlA should provide an excellent contrast with MMO. We believe that this will allow the roles of the diiron cluster, protein environment, and interactions with other components in diiron oxygenase catalysis to be investigated. The study of the CmlA may also lead to important insights into strategies for the production of novel antibiotics. PUBLIC HEALTH RELEVANCE: We propose to study the O2 activation reaction of the novel dinuclear iron cluster-containing ?-hydroxylase enzyme CmlA from the chloramphenicol biosynthetic pathway of Streptomyces. This enzyme utilzes a protein fold not previously known to support O2 activation. Moreover, the regulation of this process that prevents release of reactive oxygen species is also unique. Protein sequence comparisons suggest that CmlA is the first member of a large family of enzymes involved in antibiotic biosynthesis. The project should provide both fundamental insight into the essential processes of oxygen activation and oxygen incorporation as well as new synthetic strategies for antibiotics and natural products.